The overall goal of this project is to elucidate the ontogeny of regulation of cardiac contraction by developmental isoforms of troponin I (TnI) and troponin T (TnT). Contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin. Troponin is comprised of three components, two of which, TnI and TnT, undergo isoform switching during cardiac development. The central hypothesis of this proposal is that observed differences of contractile function comparing immature and mature heart are in part mediated by TnI and TnT developmental isoforms conferring significant effects on regulation of contraction by affecting Ca2+ sensitivity of tension, by affecting cooperative activation of thin filaments by bound cross-bridges, and by affecting cross-bridge interaction kinetics. Experiments to address the following specific aims will be performed: (Specific Aim #1) to identify TnT and TnI developmental isoforms in rat heart, (Specific Aim #2) to characterize the tension-pCa relationship during TnT and TnI isoform transitions at various developmental stages and in response to reconstitution with TnT and TnI developmental isoforms, (Specific Aim #3) to determine the effects of TnT and TnI developmental isoforms on activation of contraction by Ca2+ sensitizing agents and cooperative activation by strong binding myosin cross-bridges, and (Specific Aim #4) to determine possible roles of TnT and Tnl developmental isoforms in determining cross-bridge cycling kinetics. The relationship between TnI and TnT isoform expression (determined by SDS-PAGE, ultra-sensitive silver staining, and immunoblotting) and myofibrillar contractile behavior (tension-pCa relationship, cross-bridge cycling rate, cross-bridge number, force per cross-bridge, effect of the strong binding myosin S-1 derivative NEM-S1) will be assessed in native skinned trabeculae and single myocytes from rat heart at various developmental stages during TnT and TnT isoform transitions and in preparations in which native TnI and/or TnI is extracted and replaced with specific TnT and TnI developmental isoforms. Experiments in this proposal will elucidate specific roles of thin filament proteins TnI and TnT in the development of regulation of cardiac contraction. The applicant anticipates extending this unique multi-level approach to studies of cross-bridge kinetics during development utilizing caged compounds, to studies employing mutated TnI and TnT in order to determine site (or sites) conferring observed developmental changes, and to studies relating myofibrillar protein content and contractile dysfunction in congenital or acquired cardiomyopathies.